Engineering Coupled Consortia-Based Biosensors using Incoherent Feedforward Loops

Abstract Synthetic multicellular systems offer the potential for performing complex biological tasks through coordinated cell-to-cell communication. However, the engineering of these systems faces challenges, including managing population ratios, cell growth rates, and asynchronous activity. To minimize the impact of these factors on the multicellular design, we developed coupled bacterial consortia designed for multi-marker detection by employing a stable shared quorum sensing signal, enabling the simultaneous initiation of their activities. The shared signal is driven by an incoherent feedforward loop (IFFL) gene circuit capable of generating a consistently low-level plateau-like signal that persists over 20 cell generations. This stability ensures a consistent synthesis rate of molecules and prevents unintended amplification during signal transduction, allowing for the efficient simultaneous initiation of activities among consortium members. We examined the consortia-based biosensor by monitoring Lactate and Heme in humanized fecal samples for 40 hours. Our findings provide insights into the IFFL system's dynamic response and long-term temporal stability, making them valuable for biotechnological applications. Particularly during the scaling of synthetic multicellular systems, where maintaining consistent cell density ratios among different strains within consortia is critical.